Glial cells play myriad roles in the developing nervous system. Among other roles, they appear to be key players in the formation of compartmented neuropils in the somatosensory cortex and elsewhere in the CNS. Direct tests to determine the nature and mechanisms of glial involvement, however, are difficult in these mammalian systems. The antennal lobe of the brain of the moth Manduca sexta is an especially tractable system in which to test these roles. In our previous studies of the development of the antennal lobe indicate an essential role for glial cells in mediating interactions between ingrowing sensory axons and CNS neurons, that lead to the formation of olfactory glomeruli. In our previous studies in the antennal system, we have generated a detailed anatomical picture of neuronal development, but our understanding of glia-cells is based primarily on nuclear or cell-body staining. Recent evidence using an acute slice preparation suggests that glial cells in the antennal lobe can have complicated morphologies and, moreover, that they exhibit changing biophysical properties and changing patterns of coupling. In the proposed study, we will use the slice preparation to explore the development of glial cells in the context of the normal 3-dimensional architecture of the lobe. We will ask: 1) How does the pattern of morphological development of the neuropilar glial cells contribute to the formation of glomeruli? We will describe the morphological development of individual glial cells, and spatial relationships of glia with other glia and with specific classes of neurons. 2) Could gap junctions among glia provide a substrate for the passage of developmental signals that lead to formation of compartments: Are there regional or compartmental patterns of coupling, and do the patterns change with the arrival of sensory axons: 3) How does the arrival of sensory axons influence glial-cell differentiation? Are the morphologies and voltage-gated currents expressed by glial from normally afferented lobes different from those in lobes that have developed in the absence of sensory axons? Could A Ch, the presumed transmitter of olfactory sensory axons, act as a signal influencing the development of the glial borders? 4) Does serotonin (5-HT) influence glial differentiation? A single 5-HT-immunoreactive neuron is present in the antennal lobe throughout postembryonic development. In view of evidence from other systems that 5-HT can modulate developmental processes and induce change in glial behavior, we will ask; Do glia respond to direct application of 5-HT with a change in voltage-gated currents, a change in coupling, or a change in internal calcium? If we remove the 5-HT innervation in the lobe during glomerulus formation, will glomeruli form normally" 5) Develop a protocol to generate long-term organotypic slice cultures of developing antennal lobes and antennae. Successful completion of the aims will elucidate, in the most detail to date in any system, the involvement of glial cell in afferent-axon induced development of compartments within the CNS.